Telomeres are unique structures located at the ends of chromosomes. Their function is to prevent DNA ends from being recognized as DNA breaks which leads to activation of DNA repair mechanisms and results in cell growth arrest or death. During each cell replication cycle, a portion of the telomere is lost. Continued telomere shortening ultimately results in a growth-arrested state known as senescence or our molecular clock. Telomerase is an enzyme that elongates telomeric DNA, resulting in maintenance of telomeres and cellular immortality. Importantly, more than 85% of all malignant human cancers and almost 100% of advanced cancers express high levels of hTERT, the human catalytic subunit of telomerase.
hTERT expression has long been seen as a potential diagnostic or prognostic biomarker for cancer since activity is almost always high in cancer cells and low in normal cells. In many tumour types, the level of hTERT correlates with prognosis, and in other cases it can distinguish between malignant and benign lesions. Nevertheless, there are several flaws which make measurement of telomerase activity and/or hTERT expression difficult in the clinical setting. These include: 1) there is no reliable antibody for hTERT; 2) high-quality samples are required for accurate measurement of the entire hTERT complex (including the RNA and protein components of the enzyme) or RNA molecules, which are unstable and degradable; 3) clinical preparation of samples using formalin-fixation and paraffin-embedding (FFPE) further degrades RNA and, thus, the determination of TERT enzymatic activity and gene expression are unfeasible in most tumour samples; and 4) activated lymphocytes may have elevated hTERT expression contaminating the tumour tissue and producing false positive results. For these reasons, hTERT expression is currently not used as a clinical diagnostic and prognostic tool in cancer.
DNA methylation is the biochemical addition of a methyl group (CH3) to a nucleotide molecule, which in adult mammals occurs predominantly to cytosines in cytosine-guanosine (CpG) dinucleotides. The promoter regions of most genes, including hTERT, contain CpG-rich regions known as CpG islands and DNA methylation is an epigenetic mechanism that can modulate expression of downstream genes. In most cases, promoter hypermethylation reduces gene expression. The hTERT promoter, however, is a large, complex promoter with a poorly understood methylation profile. Specifically, hypermethylation of the same promoter region occurs in both cancer and normal tissues and is associated with variable hTERT expression. In other regions of the promoter, there is hypomethylation in both cancer and normal tissue which is associated with variable hTERT expression.
It would be desirable, thus, to develop diagnostic and/or prognostic methods for cancer that overcome one or more of the disadvantages outlined above.